Neurons communicate using electrical activity, but the exchange of information between neurons usually requires the release of chemical messengers. This fast synaptic transmission is tightly regulated by the flux of calcium ions into specialized active zone regions of the presynaptic neuron. The magnitude, timing, and spatial distribution of calcium influx determines the amount of transmitter that is released. We propose to use high speed imaging at the frog neuromuscular junction to increase our understanding of the spatial distribution of the calcium influx profiles that regulate release at this synapse. The frog neuromuscular junction is a classic synaptic preparation with large and well-organized active zones. As such, this preparation combines a spatial organization that is favorable for calcium imaging at the level of single active zones with a wealth of background information related to the physiology and anatomy of this synapse. We propose to characterize with fast temporal resolution the spatial distribution of calcium influx along the length of the neuromuscular junction. We will test the hypothesis that the opening of a single Ca2+ channel generates a stimulus-evoked Ca2+ entry site and that single channel openings trigger transmitter release in the frog neuromuscular junction. In addition, we are interested in studying the effects of G protein modulation of calcium influx through voltage-gated channels at this synapse. These studies will increase our understanding of transmitter release and its modulation.